Neurotrophic Effects of GnRH on Neurite Outgrowth and Neurofilament Protein Expression in Cultured Cerebral Cortical Neurons of Rat Embryos

The presence of GnRH receptor in cerebral cortical neurons of rat embryos and adult rats has been described. In this work, we studied the effects of GnRH on outgrowth and length of neurites and cytoskeletal neurofilament proteins expression (NF-68 and NF-200 kDa) by immunoblot of cultured cerebral cortical neurons of rat embryos. Our results show that GnRH increases both outgrowth and length of neurites accompanied by an increase in neurofilaments expression. It is conceivable that GnRH plays a role in neuronal plasticity parallel to its gonadal function.     

Colloquium 14: New Functions for Retinoic Acid in the Developing CNS. The role of retinoic acid in the patterning of the developing nervous system

Retinoic acid (RA) is metabolised from its precursor, retinol (vitamin A). In mammalian embryos, retinol is provided by the mother via the placenta and in birds retinol comes from the yolk. We have studied the role of RA in CNS development in quail embryos by depriving adult quails of retinol in the diet which results in them laying eggs which have no retinol stores. The resulting embryos are therefore retinol and RA deficient. The CNS of these embryos is abnormal in three regards; patterning, neural crest production and neurite outgrowth. With regard to patterning, at an early stage of development prior to somitogenesis, hindbrain patterning genes are not induced which leads to the respecification of the posterior hindbrain territory. This region is not lost from the embryo but instead becomes transformed into an enlarged anterior hindbrain. Another aspect of patterning that is abnormal in these RA deficient embryos is the dorsoventral gene expression domains in the anterior spinal cord. These domains are required for the proper specification of motor neurons, sensory neurons and various classes of interneurons. Consequently these neuronal classes are mis‐specified in the RA deficient embryos. With regard to the neural crest, these cells often fail to migrate correctly and then die in the absence of RA. With regard to neurite outgrowth, very little outgrowth seems to take place in these deficient embryos which suggests that RA is involved in neurite outgrowth. Taking these experiments into the adult to examine the role of RA in neurite regeneration, we have had success in inducing neurite outgrowth in vitro from adult mouse spinal cord by manipulating the retinoic acid receptors which transduce the RA signal at the level of the nucleus.     

Triad1 regulates the expression and distribution of EHD1 contributing to the neurite outgrowth of neurons after spinal cord injury

Traumatic spinal cord injury is a common and severe complication after an accident. As we all know that neurite outgrowth of neurons is difficult after a spinal cord injury. Endosome system is associated with cargoes transportation and contributes in promoting the neuronal capability for neurite outgrowth. EH domain-containing protein 1 (EHD1) transports proteins through the endosome system, especially in the recycling endosomes and regulating the neurite outgrowth. In mammalian cells, the involvement of the ubiquitin-proteasome system in endosomal sorting has been well established. Two RING fingers and a DRIL (double RING finger-linked) 1 (Triad1) plays an important role in membrane trafficking and its mutant results in the wrong accumulation of receptors in endosomes and plasma membrane. In this current study, we reasonably integrated the results of the above research and investigated the regulating function of Triad1 to EHD1 following the spinal cord injury. We characterized the upregulated expression and distribution of Triad1 and EHD1 in the neurons after SCI and declared the interaction between Triad1 with EHD1 both in vitro and in vivo. Triad1 regulated the interaction between itself and the full-length or EH domain of EHD1, which influenced the neurite outgrowth of PC12 cells. Our data delineate a novel interaction between Triad1 and EHD1 that may contribute to the regulation of neurite outgrowth for neurons after the spinal cord injury.     

Nr-CAM promotes neurite outgrowth from peripheral ganglia by a mechanism involving axonin-1 as a neuronal receptor.

Nr-CAM is a neuronal cell adhesion molecule (CAM) belonging to the immunoglobulin superfamily that has been implicated as a ligand for another CAM, axonin-1, in guidance of commissural axons across the floor plate in the spinal cord. Nr-CAM also serves as a neuronal receptor for several other cell surface molecules, but its role as a ligand in neurite outgrowth is poorly understood. We studied this problem using a chimeric Fc-fusion protein of the extracellular region of Nr-CAM (Nr-Fc) and investigated potential neuronal receptors in the developing peripheral nervous system. A recombinant Nr-CAM-Fc fusion protein, containing all six Ig domains and the first two fibronectin type III repeats of the extracellular region of Nr-CAM, retains cellular and molecular binding activities of the native protein. Injection of Nr-Fc into the central canal of the developing chick spinal cord in ovo resulted in guidance errors for commissural axons in the vicinity of the floor plate. This effect is similar to that resulting from treatment with antibodies against axonin-1, confirming that axonin-1/Nr-CAM interactions are important for guidance of commissural axons through a spatially and temporally restricted Nr-CAM positive domain in the ventral spinal cord. When tested as a substrate, Nr-Fc induced robust neurite outgrowth from dorsal root ganglion and sympathetic ganglion neurons, but it was not effective for tectal and forebrain neurons. The peripheral but not the central neurons expressed high levels of axonin-1 both in vitro and in vivo. Moreover, antibodies against axonin-1 inhibited Nr-Fc-induced neurite outgrowth, indicating that axonin-1 is a neuronal receptor for Nr-CAM on these peripheral ganglion neurons. The results demonstrate a role for Nr-CAM as a ligand in axon growth by a mechanism involving axonin-1 as a neuronal receptor and suggest that dynamic changes in Nr-CAM expression can modulate axonal growth and guidance during development.     

Explant cultures of teleost spinal cord: source of neurite outgrowth

The source of neurite outgrowth in explant cultures of normal adult Apteronotus spinal cord was examined. Explants which contained the central region of spinal cord, including ependyma, showed neurite outgrowth in culture. Explants which did not contain ependyma showed no neurite outgrowth. It is concluded that the ependymal region is necessary for neurite outgrowth in these cultures of adult teleost spinal cord. In addition, our failure to observe axon outgrowth clearly attributable to fluorescently back-labeled electromotor neurons in these cultures suggests that the exuberant neurite outgrowth in vitro is most probably due to cells other than the electromotor neurons. This explant culture system provides a unique opportunity to study neuronal differentiation, regeneration, and neurogenesis in vitro.     

CNS neurotrophins are biologically active and expressed by multiple cell types

Neutrotrophins are increasingly appreciated as potential modulators of neuronal function in the adult central nervous system (CNS). To describe the neurotrophin environment within the adult CNS, mRNA and protein expression patterns of neurotrophins-3 and –4 and of brain-derived neurotrophin were investigated in adult rat spinal cord and brain. Co-localization studies with CNS cell type-specific markers demonstrates that multiple cell types, including both neurons and glia, express these neurotrophins in the normal adult CNS. Although widely implicated in important CNS functions such as synaptic plasticity, biological activity of endogenous CNS neurotrophins has not been directly demonstrated. With a sensitive neurite outgrowth bioassay we demonstrate that CNS neurotrophins elicit neurite outgrowth and are biologically active. Moreover, antibody-blocking studies suggest that these three neurotrophins may comprise the bulk of adult CNS neurotrophic activity.     

Target-derived astroglia regulate axonal outgrowth in a region-specific manner.

The potential neuroanatomical specificity of astrocyte influence on neurite outgrowth was studied using an in vitro coculture system in which neurons from embryonic rat spinal cord or hippocampus were grown for 4 days in the presence of, but not in direct contact with, astrocytes derived either from the same region (homotopic coculture) or from different regions (heterotopic coculture) of the rat central nervous system. The results showed that axonal outgrowth was greatly enhanced in heterotopic cocultures in which spinal cord or hippocampal neurons were grown with astrocytes derived from their appropriate CNS target regions. This effect was remarkably specific, because the astroglia harvested from spinal or hippocampal target regions were not effective in promoting axon growth of nonafferent neuronal populations. Dendritic outgrowth was similar under all coculture conditions. These data suggest that diffusible signals, produced by astrocytes, can regulate neurite extension in vitro in a neuroanatomically specific manner and that axons are more sensitive than dendrites to the regional astrocyte environment.     

Studies on expression of FSH and its anti-apoptotic effects on ischemia injury in rat spinal cord

Studies indicated that many tissues could express FSH. New functions of FSH have been recognized beyond reproduction regulation. However, no report has been made about the expression and function of FSH in rat spinal cord. Double-labeled immunofluorescence stain and in situ hybridization were used to study the co-localization of FSH with its receptor and co-localization of FSH with GnRH receptor in rat spinal cord. Spinal cord ischemia injury models were built, TUNEL stain and Fas immunostaining were made to observe the anti-apoptotic effects of FSH to neurons induced by spinal cord ischemia injury. The results found that some neurons and glias of rat spinal cord showed both FSH immunoreactivity and FSH mRNA positive signals; not only FSH and its receptor but also FSH and GnRH receptor co-located in cells of both gray matter and white matter; treatment with certain concentration of FSH before ischemia–reperfusion injury, less TUNEL positive cells and Fas positive cells were found in motor neurons of ventral gray matter in FSH experiment group than that in control group. These suggested that rat spinal cord could express FSH, it is also a target organ of FSH; FSH might exert functions through its receptor by paracrine or autocrine effects; GnRH in spinal cord might regulate FSH positive neurons through GnRH receptor; FSH might inhibit ischemia induced neuron apoptosis by down-regulating Fas expression in spinal cord.     

Role of phospholipase D1 in neurite outgrowth of neural stem cells

Employing neural stem cells from the brain cortex of E12 rat embryos, we investigated the possible role of phospholipase D (PLD) in the synaptogenesis and neurite formation of neural cells during differentiation. Expression level of PLD1 increased during neuronal differentiation of the neural stem cells, resulting in increased PLD activity. Expression level of synapsin I, a marker of synaptogenesis, also increased as the differentiation of neural stem cells progressed. To figure out the effect of PLD on synapsin I expression, we treated the neural stem cells with phorbol myristate acetate (PMA) to stimulate PLD activity. Increased PLD activity induced by PMA treatment resulted in elevated synapsin I expression and neurite outgrowth during neuronal differentiation. To further confirm the role of PLD in neurite outgrowth, we transfected the dominant-negative form of rat PLD1 cDNA (DN-rPLD1) into neural stem cells to downregulate PLD activity. Overexpression of DN-rPLD1 showed the complete inhibition of neurite outgrowth of neural stem cells under differentiation condition. While transfection of DN-rPLD1 did not affect the synapsin I expression, overexpression of rPLD1 resulted in increased synapsin I expression of the neural cells. These results suggest that PLD1 plays a critical role in neurite outgrowth during differentiation of the neural stem cells. In conclusion, this is the first evidence to show that PLD1 acts as an important regulator of neurite outgrowth in neural stem cell by promoting neuronal differentiation via increase of synapsin I expression.     

tPA Regulates Neurite Outgrowth by Phosphorylation of LRP5/6 in Neural Progenitor Cells

Despite the important role of tissue plasminogen activator (tPA) as a neuromodulator in neurons, microglia, and astrocytes, its role in neural progenitor cell (NPC) development is not clear yet. We identified that tPA is highly expressed in NPCs compared with neurons. Inhibition of tPA activity or expression using tPA stop, PAI-1, or tPA siRNA inhibited neurite outgrowth from NPCs, while overexpression or addition of exogenous tPA increased neurite outgrowth. The expression of Wnt and β-catenin as well as phosphorylation of LRP5 and LRP6, which has been implicated in Wnt–β-catenin signaling, was rapidly increased after tPA treatment and was decreased by tPA siRNA transfection. Knockdown of β-catenin or LRP5/6 expression by siRNA prevented tPA-induced neurite extension. NPCs obtained from tPA KO mice showed impaired neurite outgrowth compared with WT NPCs. In ischemic rat brains, axon density was higher in the brains transplanted with WT NPCs than in those with tPA KO NPCs, suggesting increased axonal sprouting by NPC-derived tPA. tPA-mediated regulation of neuronal maturation in NPCs may play an important role during development and in regenerative conditions.     

Evaluation of the role of Disabled-2 in nerve growth factor-mediated neurite outgrowth and cellular signalling

Disabled-2 (DAB2) is an adapter protein that plays a key role in cell proliferation and differentiation. We reported here that DAB2 is expressed in various regions of rat central nervous system and is most abundant in the olfactory bulb. The up-regulation of DAB2 upon 5,7-dihydroxytryptamine-induced spinal cord lesion implicates that DAB2 may participate in the regulation of neuronal plasticity. To investigate DAB2 function in the regulation of neurite outgrowth, the rat p59 and p82 form of DAB2 was individually and stably expressed in the PC12 cells. Both p59 and p82 inhibited nerve growth factor (NGF)-induced neurite outgrowth concomitantly with a decrease in the expression of neuron-specific cytoskeleton protein beta-tubulin III. To unveil the molecular mechanism of DAB2 in NGF signaling, we found that RhoA-GTPase activity was up-regulated in DAB2 stable lines whereas the Ras/MAPK and PI3-kinase/Akt signaling was not affected. The inhibitory effect of DAB2 on NGF-mediated neurite outgrowth was reversed by the pretreatment of Rho-kinase (ROCK) inhibitor Y27632, implicating that DAB2 modulates RhoA/ROCK signaling. Together, this study defines a role of DAB2 in the control of neuronal plasticity and demonstrates for the first time that DAB2 is a negative regulator in NGF-mediated neurite outgrowth.     

Role of spastin and protrudin in neurite outgrowth

Hereditary spastic paraplegia (HSP) is a neurodegenerative disorder characterized by retrograde axonal degeneration that primarily affects long spinal neurons. The gene encoding spastin has a well-established association with HSP, and protrudin is a known binding partner of spastin. Here, we demonstrate that the N-terminal domain of protrudin mediates the interaction with spastin, which is responsible for neurite outgrowth. We show that spastin promotes protrudin-dependent neurite outgrowth in PC12 cells. To further confirm these physiological functions in vivo, we microinjected zebrafish embryos with various protrudin/spastin mRNA and morpholinos. The results suggest that the spinal cord motor neuron axon outgrowth of zebrafish is regulated by the interaction between spastin and protrudin. In addition, the putative HSP-associated protrudinG191V mutation was shown to alter the subcellular distribution and impair the yolk sac extension of zebrafish, but without significant defects in neurite outgrowth both in PC12 cells and zebrafish. Taken together, our findings indicate that protrudin interacts with spastin and induces axon formation through its N-terminal domain. Moreover, protrudin and spastin may work together to play an indispensable role in motor axon outgrowth.     

Repulsive guidance molecule b inhibits neurite growth and is increased after spinal cord injury

Neuronal axons are guided by attractive and repulsive cues in their local environment. Since the identification of the repulsive guidance molecule (RGM) a (RGMa) as an axon repellent in the visual system, diverse functions, as part of the developing and adult central nervous system (CNS), have been ascribed to it. The binding of RGMa to its receptor neogenin has been shown to induce RhoA activation, leading to inhibitory/repulsive behavior and the collapse of the neuronal growth cone. In this paper, we provide evidence to suggest the involvement of RGMb, another member of the RGM family, in the rat CNS. RGMb inhibits neurite outgrowth in postnatal cerebellar granule neurons (CGNs) in vitro. RGMb is expressed by oligodendrocytes and neurons in the adult rat CNS, and the expression of this molecule is upregulated around the site of spinal cord injury. RGMb is present in myelin isolated from an adult rat brain. RGMb and neogenin are coexpressed in CGNs and entorhinal cortex neurons. These findings suggest that RGMb is a myelin-derived inhibitor of axon growth in the CNS. Inhibition of RGMb may provide an alternative approach for the treatment of spinal injuries.     

Permissive role of Cdc2 activity induced from astrocytes in neurite outgrowth

Following spinal cord injury, glial cells are recognized as major environmental factors hampering axon's regenerative responses. However, recent studies suggested that, in certain circumstances, reactive astrocytes may have a permissive role for axonal regeneration and functional recovery. Here, we report that Cdc2 activation in astrocytes is positively linked to axon growth. Cdc2 was strongly, but transiently, induced from reactive astrocytes within and around the injury cavity. Cdc2 levels in primary, non‐neuronal cells prepared from injured spinal cord were up‐regulated by extending the pre‐injury period. Cdc2‐mediated vimentin phosphorylation was strongly induced in astrocytes after long‐term culture (7 days, LTC) as compared with short‐term culture (3 days, STC). Induction levels of phospho‐vimentin in LTC astrocytes were positively associated with increased neurite outgrowth in co‐cultured dorsal root ganglion neurons. β3 integrin mRNA was induced in LTC astrocytes and activation of β3 integrin was regulated by Cdc2 activity. Furthermore, genetic depletion and pharmacological blockade experiments demonstrate that activation of Cdc2 and β3 integrin in LTC astrocytes is required for neurite outgrowth. Our data suggest that the Cdc2 pathway may play an important role in determining phenotypic expression of astrocytes such that astrocytes provide permissive environments for axonal regeneration following spinal cord injury.     

Glutamate regulates neurite outgrowth of cultured descending brain neurons from larval lamprey

In larval lamprey, descending brain neurons, which regenerate their axons following spinal cord injury, were isolated and examined in cell culture to identify some of the factors that regulate neurite outgrowth. Focal application of 5 mM or 25 mM L-glutamate to single growth cones inhibited outgrowth of the treated neurite, but other neurites from the same neuron were not inhibited, an effect that has not been well studied for neurons in other systems. Glutamate-induced inhibition of neurite outgrowth was abolished by 10 mM kynurenic acid. Application of high potassium media to growth cones inhibited neurite outgrowth, an effect that was blocked by 2 mM cobalt or 100 microM cadmium, suggesting that calcium influx via voltage-gated channels contributes to glutamate-induced regulation of neurite outgrowth. Application of glutamate to growth cones in the presence of 2 microM omega-conotoxin MVIIC (CTX) still inhibited neurite outgrowth, while CTX blocked high potassium-induced inhibition of neurite outgrowth. Thus, CTX blocked virtually all of the calcium influx resulting from depolarization. To our knowledge, this is the first direct demonstration that calcium influx via ligand-gated ion channels can contribute to regulation of neurite outgrowth. Finally, focal application of glutamate to the cell bodies of descending brain neurons inhibited outgrowth of multiple neurites from the same neuron, and this is the first demonstration that multiple neurites can be regulated in this fashion. Signaling mechanisms involving intracellular calcium, similar to those shown here, may be important for regulating axonal regeneration following spinal cord injury in the lamprey.     

The Promotive Effects of Thymosin β4 on Neuronal Survival and Neurite Outgrowth by Upregulating L1 Expression

Thymosin β₄ (Tβ4) is a major actin-sequestering peptide widely distributed in mammalian tissues including the nervous system. The presence of this peptide in the nervous system likely plays a role in synaptogensis, axon growth, cell migration, and plastic changes in dendritic spine. However, the effects of Tβ4 on the survival of neurons and axonal outgrowth have still not been fully understood. So far it is not clear if the effects of Tβ4 are associated with L1 functions. In the present study, we hypothesized that Tβ4-induced up-regulation of L1 synthesis could be involved in the survival and axon outgrowth of cultured spinal cord neurons. To test this hypothesis, primarily cultured neurons were prepared from the mouse spinal cord and treated with various concentrations of Tβ4 ranging from 0.1 to 10 μg/ml. The analysis of L1 mRNA expression and protein synthesis in neurons was then carried out using RT-PCR and western blot assays, respectively. After the addition of Tβ4 to cultures, cells were then treated with antibodies against distinct domains of L1-Fc. Subsequently, β-tubulin III and L1 double-labeled indirect immunofluorescence was carried out. Meanwhile, L1 immunofluorescent reactivity was analyzed and compared in cells treated with Tβ4. Furthermore, the number of β-tubulin III-positive cells and neurite lengths were measured. We found that Tβ4 enhanced L1 expression in a dose-dependent manner, and the highest L1 mRNA and protein synthesis in cells increased by more than 2.1- and 2.3-fold in the presence of Tβ4 at identical concentrations, respectively. Moreover, it also dose dependently enhanced neurite outgrowth and neuronal survival. Compared to conditions without Tβ4, the length of neurite and neuronal survival increased markedly in presence of 0.5, 1, and 5 μg/ml Tβ4, respectively, whereas the effects of Tβ4 were significantly attenuated or inhibited in the process of L1-Fc antibodies treatment. These above results indicate that the promotive effect of Tβ4 on the survival and neurite outgrowth of cultured spinal cord neurons might be mediated, at least in part via a stimulation of the production of L1 in the neurons.     

RGMa inhibition promotes axonal growth and recovery after spinal cord injury

Repulsive guidance molecule (RGM) is a protein implicated in both axonal guidance and neural tube closure. We report RGMa as a potent inhibitor of axon regeneration in the adult central nervous system (CNS). RGMa inhibits mammalian CNS neurite outgrowth by a mechanism dependent on the activation of the RhoA-Rho kinase pathway. RGMa expression is observed in oligodendrocytes, myelinated fibers, and neurons of the adult rat spinal cord and is induced around the injury site after spinal cord injury. We developed an antibody to RGMa that efficiently blocks the effect of RGMa in vitro. Intrathecal administration of the antibody to rats with thoracic spinal cord hemisection results in significant axonal growth of the corticospinal tract and improves functional recovery. Thus, RGMa plays an important role in limiting axonal regeneration after CNS injury and the RGMa antibody offers a possible therapeutic agent in clinical conditions characterized by a failure of CNS regeneration.     

Developmental and spatial expression pattern of syntaxin 13 in the mouse central nervous system

Vesicular transport involves SNARE (soluble- N-ethylmaleimide-sensitive-factor-attachment-protein-receptor) proteins on transport vesicles and on target membranes. Syntaxin 13 is a SNARE enriched in brain, associated with recycling endosomes; its overexpression in PC12 cells promotes neurite outgrowth. This suggests an important role for receptor recycling during neuronal differentiation. Here we describe the spatiotemporal pattern of syntaxin 13 expression during mouse brain development. During early embryogenesis (E12-E15), it was found in the forebrain ventricular zone and in primary motor and sensory neurons in the brainstem, spinal cord and sensory ganglia. In the forebrain at E15, syntaxin 13 was not detected in neuroblasts in the intermediate zone of the embryonic hemispheric wall, while there was labeling in cortical neurons in deeper layers starting at E15-18, and progressively in later-generated neurons up to layer II around P6. Syntaxin 13 reached maximal expression in all brain divisions at about P7, followed by a decrease, with heterogeneous neuron populations displaying various staining intensities in adult brain. While usually restricted to the soma of neurons, we transiently detected syntaxin 13 in dendrites of pyramidal neurons during the first postnatal week. In conclusion, the developmentally regulated syntaxin 13 expression in various neuronal populations is consistent with its involvement in endocytic trafficking and neurite outgrowth.     

Requirement of N-myc protein down-regulation for neuronal differentiation in the spinal cord

Previous work has indicated that N-myc expression occurs widely in the developing central nervous system, but its level changes dynamically with region- and stage-specificities. We show in the present report that in the developing spinal cord of the mouse, N-myc protein expression takes place in the ventricular zone and reaches its maximum at the outermost layer, but is extinct in the intermediate zone, indicating that N-myc protein is not expressed in mature neurons. We examined the effect of forced, persistent N-myc expression in development of the spinal cord in order to understand the functional significance of N-myc down-regulation. We made embryonic stem (ES) cell lines that constitutively expressed N-myc at a high level, then produced mouse embryo chimeras with a high contribution of the ES cells. The majority of the chimeras developed to day 12 with normal gross morphology, but in these chimeras neuronal differentiation in the spinal cord was perturbed at the histological level. Intermediate zones and ventral horns were formed, but the expression of N-CAM and neurofilaments was diminished. Chimeras using β-galactosidase-expressing recipient embryos indicated that inhibition of the neuronal differentiation was a cell-autonomous effect of persistent N-myc expression. These observations indicate that N-myc down-regulation in individual cells is required for full differentiation of neurons.